Field of the Invention
The invention relates to the preparation of samples on mass spectrometric sample supports with dispensing of liquids, and particularly to devices and methods to clean the dispenser.
Description of the Related Art
The preparation of samples for ionization by matrix-assisted laser desorption (MALDI) or similar ionization methods requires the use of organic solvents to apply the solution containing matrix material onto the individual samples under analysis. These samples have to be applied to the sample support manually or with the aid of pipetting robots. The solvents must subsequently be vaporized in order to allow crystals of the matrix substance, into which the analyte substances have to be embedded, to grow. Since ionization by matrix-assisted laser desorption (MALDI) and its requirements are widely known, no detailed description will be given here.
Today, ionization by matrix-assisted laser desorption is used widely for the mass spectrometric identification of microbes. This identification of microbial samples involves the daily preparation of hundreds of thousands of samples in many hundreds of microbiological laboratories. The preparation of microbes should serve here as an example of sample preparation.
Since only the substances from inside the microbe cells are usable for the mass spectrometric identification, the microbe cells must first be cell disrupted. This cell disruption also takes place predominantly on the sample support. The first step is to apply small, hardly visible quantities of around 105 to 107 microbes from agar plate colonies onto the test sites of the sample support. This transfer of microbes is widely done manually, but there are automatic devices available for this purpose. The cells of the microbes are usually cell disrupted on the sample support by strong acids, which must subsequently be dried up by vaporization. The acids used for this purpose are 70-percent formic acid (boiling point 101° C.; vapor pressure 43 hPa at 20° C.) or trifluoroacetic acid of similar concentration (TFA; boiling point 72° C.; vapor pressure 110 hPa at 20° C.). Quantities of around one microliter are applied onto each sample. When they have dried, the matrix solution is applied, also in quantities of around one microliter. The matrix solution usually contains a solid organic acid (usually α-cyano-4-hydroxycinnamic acid, HCCA, but also 2,5-dihydroxybenzoic acid, DHB, for example) in a solvent mixture of acetonitrile and alcohols. For reasons of occupational health and safety, ethanol is usually used, although methanol would be the better alternative. If the cell walls have not yet been completely destroyed by the acid, the matrix solution penetrates into the microbes through the weakened cell walls and causes them to burst by osmosis. Soluble contents, in particular the soluble proteins, then dissolve in the matrix solution. The drying of the matrix solution causes tiny crystals of matrix substance to form, into whose crystal lattice or crystal boundaries molecules of the contents are embedded. The microbes are then identified with the aid of a mass spectrum of the contents.
The sample supports are usually the size of microtitration plates (or a fraction thereof) and nowadays usually have 48, 96 or 384 visible test sites for the application and preparation of the samples. Sample supports with 1536 test sites are also in use. The test sites with diameters of 0.8 to 3.0 millimeters can be identified in some embodiments with the aid of milled-in rings, whose sharp milled edges prevent the applied acids and solvents from flowing laterally away. The test sites can also take the form of hydrophilic areas in a hydrophobic environment.
The sample support can especially also contain small pins, around 2 millimeters in diameter, which are inset into the sample support so as to be flush with the surface. The pins can be individually loaded with microbes by direct contact with microcolonies on agar surfaces. This can greatly shorten the culturing times, as is disclosed in the patent application WO 2013/182648 A1 assigned to the Applicant. The holes for the pins can have a slight chamfer, which keeps the edges of the pins clear so that the surface tension of the liquid applied to the end of the pins prevents it from running over the edge of the pins.
At present, the preparation is largely carried out manually with dispensing pipettes, without a hood, because hoods are rare in microbiological laboratories. This can be a health hazard if the ventilation is insufficient. Even when a hood is available, it is often not used because accurate pipetting onto a small sample spot in an open hood is very awkward. So, devices for automatic preparation which automate the application of the acids and the matrix solutions, and preferably do not release hazardous vapors so that a hood is not required, are desirable.
Liquids in quantities of around one microliter do not drip by their own weight even from very fine pipette tips, but are applied according to the prior art by dabbing them onto the sample. A new pipette tip must be used for each sample in order to prevent samples being transferred. Non-contact application of the liquids onto the samples is particularly advantageous because it eliminates the need to replace the pipette tips each time.
The generation of free-flying droplets with volumes of only a few nanoliters is known particularly from printing technology; some systems operate with piezo technology, others with vapor-bubble technology. With this technology, the droplets are ejected from nozzles. This technology does not lend itself to the application described here because almost a thousand tiny droplets, whose surface area is large relative to their volume, would have to be applied to a test site, preferably without any evaporation at all. Since matrix solutions near the saturation limit have to be used, there is a risk that deposits will form on the nozzle outlets at an early stage, meaning that the drops no longer leave the nozzle in the correct direction, and that the matrix solution will crystallize out prematurely in the flying droplets.
Dispensers which apply liquids with volumes of around one microliter onto test sites positioned below the dispenser, and which operate with falling drops of correct size without direct contact of the dispenser tip to the sample site, are also known in principle and can be used here. For this type of non-contact application, it is necessary to position the dispenser exactly vertically above the test site (or the test site on the sample support vertically below the dispenser). For the dispensing of these small quantities of liquid onto the test sites, there are different technical solutions, such as detaching the drop by pressure surges in the liquid feed, acoustic shock waves generated by piezo crystals, or sudden vertical movement of the capillary tip. Particularly simple and low-cost is a dispenser unit which has outer gas channels, arranged symmetrically around the central capillary and pointing to the tip of the central capillary. There may be a single annular channel by a concentric capillary around the dispensing capillary, or there may be an arrangement of at least two, preferably three or four single channels. A tiny pump presses a drop of around one microliter out of the central capillary; a small pressure surge of air or other suitable gas through the surrounding gas channel(s) then strips the hanging drop of dispensing liquid from the central capillary and causes it to fall vertically onto the test site. A simple arrangement is shown in FIG. 1, showing two concentric capillaries, the inner for the dispensing liquid, the outer for the gas surge. The pressure surge of the gas, the height of the fall and the fall speed of the droplet must be small enough so that the drop does not splatter, but large enough so that the drop does not simply roll away on the sample surface. The non-contact deposition of the liquid drops means that replaceable pipette tips are no longer required for the preparation.
Because quite often matrix solutions near saturation have to be used to prepare the samples for a mass spectrometric analysis, the function of the dispenser is repeatedly disturbed by crystals of matrix material formed and growing at the tip of the dispenser over time. Quite often wet crystals form at the outside (or outer circumference) of the tip, creeping slowly upwards at one side of the tip during subsequent dispensing cycles. When such obstruction exists, there is a danger that the drop of dispensing liquid, prior to being detached, is drawn upwards at one side of the tip by the easy wettability of the deposit in contrast to the commonly low wettability of the tip material. As a consequence, the drop stripped off by the gas surge will not fall just vertically and eventually could miss the sample site on the sample support. According to the prior art, the dispenser has to be dismounted and cleaned frequently; for that purpose, at least the inner capillary has to be emptied, then filled with washing fluid (in most cases a pure liquid solvent). The tip has to be washed by rinsing inside and outside. Then the washing fluid is removed from the tip, and dispensing fluid has to be refilled.
Dispensers can be equipped with drop size regulation systems, likewise known since long (see, for instance, publication JP 1986-231461; Apr. 5, 1985; F. Sugaya, describing a photometric drop size measuring and regulating system; also U.S. Pat. No. 5,601,980 A or US 2006/0144331 A1).
The present disclosure references ionization by matrix-assisted laser desorption (MALDI), where ions are produced during the desorption by pulsed laser beams. It goes without saying that sample preparations for other types of ionization shall also be possible where, for example, the analyte substances in the prepared samples are first transferred into the gaseous phase, and only then ionized. Simple laser desorption in combination with chemical ionization (LDCI) can be carried out, for example, as can direct electrospray ionization from the surface (DESI), but other types of ionization can also be used. Accordingly, the term “ionization with matrix-assisted laser desorption” must not be understood as a restriction.